ABOVE: Researchers found that cancer-associated fibroblasts (CAF) influence tumor pathology by vesicle-mediated protein transfer to stromal cells. The image shows CAF in purple, endothelial cells in green, and monocytes in orange. Alice Santi

If cancer cells are a tumor’s building blocks, fibroblasts are the architects. Cancer-associated fibroblasts (CAF) coordinate tumor progression by secreting growth factors, suppressing the immune system, and remodeling the extracellular matrix. They fuel cancer cells by handing over entire mitochondria and supplying a steady stream of proteins that make tumors more invasive.1,2

For instance, CAF pass along proteins such as galectin-1, which spurs cancer cells to migrate to new tissues.3 However, researchers did not know whether fibroblasts control the behavior of other cells that make up tumors. Recently, a paper published in Science Signaling revealed that CAF taken from breast cancer cells dispatch potentially behavior-altering proteins to endothelial cells—stromal cells that line tumor blood vessels—using a specific type of vesicle.4  The work helps researchers understand communication pathways between different tumor cells and point to new therapeutic strategies.

“[It is] interesting work that further highlights the complex intercellular crosstalk between stromal cells in cancer,” said Giulia Biffi, a cancer biologist at the University of Cambridge who was not involved in the study. 

Endothelial cells aid tumors in forming the blood vessels that transport nutrients to tumor cells. But they are also the conduit for cancer drugs, said cancer researcher and study coauthor Alice Santi, who now works at the University of Florence. So, Santi and her colleagues at the Cancer Research UK Scotland Institute, where she previously worked as a postdoctoral researcher, investigated how their function is influenced by surrounding cells. 

To determine whether fibroblasts ferry proteins to endothelial cells, the researchers isolated CAF from people with breast cancer and tagged all the proteins with a fluorescent dye. When they cultured these alongside endothelial cells taken from people without cancer, the endothelial cells began to glow, pointing to protein transfer between the two cell types. When the experiment was reversed however, CAF failed to light up, suggesting one-way delivery. 

CAF release multiple types of vesicles, with varying carrying capacities. The team found that most of the CAF proteins were transferred in matrix-bound vesicles (MBV), which roll along the floor of the matrix. Compared to other vesicles that float like bubbles through tissue fluid, MBV are efficient delivery systems that can carry a heavier protein load. 

Because floating vesicles are easily collected from the cell culture medium, while MBV require detaching from the dish, some researchers may have missed clues of intercellular protein transfer, said Santi. “There are not many papers studying MBV. Most of the time they are overlooked and not taken into account.”

To confirm that protein transfer isn’t limited to cultured cells, the team injected cancer cells into a mouse that expresses a red protein on CAF. Three weeks later, they removed tumors from the animals and found red fluorescence in tumor endothelial cells, validating the mechanism in living organisms. 

Next, the team looked into the functions of secreted proteins. Mass spectrometry experiments revealed that many of the secreted proteins contribute to cell movement and adhesion, supporting the theory that CAF proteins may help endothelial cells form blood vessels within tumors. Other hits turned up proteins involved in the immune response.

The most frequently transferred protein was Thy-1 membrane glycoprotein (THY1), a cell surface antigen capable of binding both cancer cells and immune cells. Wondering whether THY1 helps endothelial cells latch onto white blood cells, the researchers cocultured the cells with CAF that lacked the THY1 gene. Significantly fewer monocytes stuck to endothelial cells when they grew alongside CAF lacking THY1 than wild type cells. 

Probing the endothelial function of THY1 in vivo, the group injected breast cancer cells and CAF into mice, along with snippets of DNA that silence expression of THY1. When they stained for CD11b, a molecule found on the surface of leukocytes, they detected higher amounts in the blood and less in the tissue in mice without THY1. The findings suggest that transfer of THY1 from CAF to endothelial cells helps immune cells to bind and infiltrate tumors.  

At this stage, however, it is unclear whether enhanced binding of endothelial cells to immune cells promotes tumor progression. Santi and her colleagues speculate that by manipulating the immune microenvironment—a key factor influencing a person’s response to cancer therapy—CAF may help tumors evade treatment. 

Next, the team investigated what type of CAF is involved in protein transfer. CAF come in two main types: inflammatory CAF (iCAF), which secrete proinflammatory cytokines, and myofibroblast CAF (myCAF), which churn out the extracellular matrix and can suppress the immune response. By searching for markers unique to the two subtypes, the researchers found that myCAF performed the bulk of the protein transfer. 

Teasing apart their specific functions is crucial for developing appropriate treatments, Santi said. 

Fibroblasts can switch between phenotypes, and the methods used by scientists to isolate and culture the cells preferentially select myofibroblasts over their inflammatory counterparts, explained Giffi. “Whether [the in vitro findings] represent the in vivo situation will need further investigation,” she added. 

References

  1. Ippolito L, et al. Cancer-associated fibroblasts promote prostate cancer malignancy via metabolic rewiring and mitochondrial transfer. Oncogene. 2019; 38(27):5339-5355.
  2. Santi A, et al. Cancer associated fibroblasts transfer lipids and proteins to cancer cells through cargo vesicles supporting tumor growth. Biochim Biophys Acta. 2015; 1853(12):3211-23.
  3. Toti A, et al. Activated fibroblasts enhance cancer cell migration by microvesicles-mediated transfer of Galectin-1. J Cell Commun Signal. 2021; 15(3):405-419.
  4. Santi A, et al. Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function. Sci Signal. 2024; 17;827.